» Enalaprilat Injection is a sterile solution of enalaprilat in a suitable vehicle for injection. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of enalaprilat (C18H24N2O5).

Packaging and storagePreserve in single-dose or in multiple-dose containers, and store at controlled room temperature.

A: The retention time of the enalaprilat peak in the chromatogram of the Assay preparation corresponds to that of the corresponding peak in the chromatogram of the Standard preparation, obtained as directed in the Assay.

Stock solution CDissolve accurately weighed quantities of benzyl alcohol, benzaldehyde, and benzoic acid in Diluent. Dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of 0.1 mg per mL of each of the three substances.

System suitability solutionPipet 4.0 mL of Stock solution A, 5 mL of Stock solution B, and 5 mL of Stock solution C into a 25-mL volumetric flask, and dilute with Diluent to volume to obtain a solution containing about 0.0032 mg per mL of enalaprilat related impurity A, 0.01 mg per mL of enalapril, and 0.02 mg per mL of each of benzyl alcohol, benzaldehyde, and benzoic acid, respectively.

Test solutionTransfer an accurately measured volume of Injection, equivalent to about 12.5 mg of enalaprilat, to a 25-mL volumetric flask. Dilute with Diluent to volume, and mix.

Diluted test solutionDilute the Test solution (1 in 100) with Diluent to obtain a solution having a known concentration of 0.005 mg per mL (corresponds to 1% of the Test solution).

Chromatographic system (see Chromatography 621)The chromatograph is equipped with 215-nm detector and a 4.6-mm × 15-cm, 5-µm column that contains packing L1. The column temperature is maintained at 60. The flow rate is about 1.5 mL per minute. The chromatograph is programmed as follows:

Time (minutes)

Solution A(%)

Solution B (%)

Elution

05

97.0

3.0

isocratic

520

97®77.5

3®22.5

linear gradient

2025

77.5®10

22.5®90

linear gradient

2525.01

10®97

90®3.0

step gradient

25.0130

97

3.0

re-equilibrium

Chromatograph the System suitability solution and the Diluted test solution, and record the peak responses as directed for Procedure: the resolution, R, between enalapril maleate and enalaprilat related impurity A is not less than 1.2; the capacity factor for enalaprilat is not less than 1.5; and the relative standard deviation of the enalaprilat peak in replicate injections of the Diluted test solution is not more than 2.0%.

ProcedureSeparately inject equal volumes (about 20 µL) of the Diluted test solution and the Test solution into the chromatograph, record the chromatogram, and measure the peak response for enalaprilat in the Diluted test solution and all of the peak responses from the Test solution that do not correspond to enalaprilat, benzyl alcohol, benzoic acid, benzaldehyde, and benzyl alcohol related compounds (see Table 1 for RRT). Calculate the percentage of specified and unspecified impurities using the formula:

(ri / rS)

in which ri is the peak response for each specified impurity in the Test solution; and rS is the peak response for enalaprilat in the Diluted test solution: the impurities meet the specified limits in Table 1.

Table 1

Component

Relative Retention Time (minutes)

Limit (w/w, %)

Enalaprilat heat degradation product

0.6 vs enalaprilat

0.5

Enalaprilat

1 vs enalaprilat



Benzyl alcohol

1 vs benzyl alcohol



Benzyl alcohol related unknown impurity 1

1.2 vs benzyl alcohol



Benzoic acid

1.4 vs benzyl alcohol



Benzyl alcohol related unknown impurity 2

1.7 vs benzyl alcohol



Benzaldehyde

2.1 vs benzyl alcohol



Enalapril maleate

4.7 vs enalaprilat

0.25

Enalaprilat related impurity A

5.1 vs enalaprilat

1.0

Any other unspecified individual impurity



0.10

Total impurities



2.0

Benzyl alcohol content (if present)

Buffer solution, Mobile phase, System suitability solution, and Chromatographic systemProceed as directed in the Assay.

Standard solutionDissolve an accurately weighed quantity of USP Benzyl Alcohol RS in Buffer solution to obtain a solution having a known concentration of 0.72 mg per mL.

Test solutionUse the Assay preparation, prepared as directed in the Assay.

ProcedureProceed as directed in the Assay. Calculate the percentage of benzyl alcohol, based on the label claim, in the volume of Injection taken by the formula:

100(CS / CU)(rU / rS)

in which CS is the concentration, in mg per mL, of USP Benzyl Alcohol RS in the Standard solution; CU is the concentration, in mg per mL, of benzyl alcohol in the Test solution; and rU and rS are the benzyl alcohol peak responses obtained from the Test solution and the Standard solution, respectively: between 75.0% and 120.0% of the labeled amount is found.

Buffer solutionPrepare a solution of 0.05 M monobasic potassium phosphate. Adjust with phosphoric acid to a pH of 2.5.

Mobile phasePrepare a filtered and degassed mixture of Buffer solution and acetonitrile (84 : 16). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability preparationUse the Standard preparation.

Standard preparationDissolve an accurately weighed quantity of USP Enalaprilat RS and USP Benzyl Alcohol RS in Buffer solution, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.1 mg per mL of enalaprilat and 0.72 mg per mL of benzyl alcohol.

Assay preparationTransfer an accurately measured volume of Injection, equivalent to about 5 mg of enalaprilat, to a 50-mL volumetric flask. Dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 215-nm and 258-nm detector (use 215 nm as the initial wavelength, and switch to 258 nm after the elution of enalaprilat and before the elution of benzyl alcohol) and a 4.6-mm×15-cm, 5-µm column that contains packing L1. The flow rate is about 1.5 mL per minute. The column temperature is maintained at 60. Chromatograph the System suitability preparation, and record the peak responses as directed for Procedure: the resolution, R, between benzyl alcohol and enalaprilat is not less than 3.0; the tailing factor for benzyl alcohol and the enalaprilat peaks is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.

ProcedureSeparately inject equal volumes (about 20 mL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure all of the peak responses. Calculate the quantity, in percentage of the label claim, of enalaprilat (C18H24N2O5) in the portion of Injection taken by the formula:

100(CS / CU)(rU / rS)

in which CS is the concentration, in mg per mL, of USP Enalaprilat RS in the Standard preparation; CU is the concentration, in mg per mL, of enalaprilat in the Assay preparation, based on the label claim; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.